This invention is in the area of biologically active nucleosides, and specifically includes antiviral compositions that include 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (xe2x80x9cFTCxe2x80x9d), its physiologically acceptable derivative, or physiologically acceptable salt, and a method for the resolution and use of the (xe2x88x92)-xcex2-L and (+)-xcex2-D enantiomers of FTC.
In 1981, acquired immune deficiency syndrome (AIDS) was identified as a disease that severely compromises the human immune system, and that almost without exception leads to death. In 1983, the etiological cause of AIDS was determined to be the human immunodeficiency virus (HIV). By December of 1990, the World Health Organization estimated that between 8 and 10 million people worldwide were infected with HIV, and of that number, between 1,000,000 and 1,400,000 were in the U.S.
In 1985, it was reported that the synthetic nucleoside 3xe2x80x2-azido-3xe2x80x2-deoxythymidine (AZT) inhibits the replication of human immunodeficiency virus. Since then, a number of other synthetic nucleosides, including 2xe2x80x2,3xe2x80x2-dideoxyinosine (DDI), 2xe2x80x2,3xe2x80x2-dideoxycytidine (DDC), 3xe2x80x2-fluoro-3xe2x80x2-deoxythymidine (FLT), and 2xe2x80x2,3xe2x80x2-dideoxy-2xe2x80x2,3xe2x80x2-didehydrothymidine (D4T), have been proven to be effective against HIV. A number of other 2xe2x80x2,3xe2x80x2-dideoxynucleosides have been demonstrated to inhibit the growth of a variety of viruses in vitro. It appears that, after cellular phosphorylation to the 5xe2x80x2-triphosphate by cellular kinases, these synthetic nucleosides are incorporated into a growing strand of viral DNA, causing chain termination due to the absence of the 3xe2x80x2-hydroxyl group.
The success of various 2xe2x80x2,3xe2x80x2-dideoxynucleosides in inhibiting the replication of HIV in vivo or in vitro has led a number of researchers to design and test nucleosides that substitute a heteroatom for the carbon atom at the 3xe2x80x2-position of the nucleoside. Norbeck, et al., disclose that (xc2x1)-1-[(2xcex2,4xcex2)-2-(hydroxymethyl)-4-dioxolanyl]thymine (referred to as (xc2x1)-dioxolane-T) exhibits a modest activity against HIV (EC50 of 20 xcexcm in ATH8 cells), and is not toxic to uninfected control cells at a concentration of 200 xcexcM. Tetrahedron Letters 30 (46), 6246, (1989). European Patent Application Publication No. 0 337 713 and U.S. Pat. No. 5,041,449, assigned to IAF BioChem International, Inc., disclose 2-substituted-4-substituted-1,3-dioxolanes that exhibit antiviral activity.
U.S. Pat. No. 5,047,407 and European Patent Application Publication No. 0 382 526, also assigned to IAF Biochem International, Inc. disclose a number of 2-substituted-5-substituted-1,3-oxathiolane nucleosides with antiviral activity, and specifically report that the racemic mixture (about the C4xe2x80x2-position) of the C1xe2x80x2-xcex2 isomer of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as (xc2x1)-BCH-189) has approximately the same activity against HIV as AZT, and no cellular toxicity at the tested levels. (xc2x1)-BCH-189 has also been found to inhibit the replication of AZT-resistant HIV isolates in vitro from patients who have been treated with AZT for longer than 36 weeks.
Another virus that causes a serious human health problem is the hepatitis B virus (referred to below as xe2x80x9cHBVxe2x80x9d). HBV is second only to tobacco as a cause of human cancer. The mechanism by which HBV induces cancer is unknown, although it is postulated that it may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis, and cell regeneration associated with the infection.
After a two to six month incubation period in which the host is unaware of the infection, HBV infection can lead to acute hepatitis and liver damage, that causes abdominal pain, jaundice, and elevated blood levels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly progressive, often fatal form of the disease in which massive sections of the liver are destroyed.
Patients typically recover from acute hepatitis. In some patients, however, high levels of viral antigen persist in the blood for an extended, or indefinite, period, causing a chronic infection. Chronic infections can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries. By mid-1991, there were approximately 225 million chronic carriers of HBV in Asia alone, and worldwide, almost 300 million carriers. Chronic persistent hepatitis can cause fatigue, cirrhosis of the liver, and hepatocellular carcinoma, a primary liver cancer.
In western industrialized countries, high risk groups for HBV infection include those in contact with HBV carriers or their blood samples. The epidemiology of HBV is very similar to that of acquired immune deficiency syndrome, which accounts for why HBV infection is common among patients with AIDS or AIDS-related complex. However, HBV is more contagious than HIV.
A human serum-derived vaccine has been developed to immunize patients against HBV. While it has been found effective, production of the vaccine is troublesome because the supply of human serum from chronic carriers is limited, and the purification procedure is long and expensive. Further, each batch of vaccine prepared from different serum must be tested in chimpanzees to ensure safety. Vaccines have also been produced through genetic engineering. Daily treatments with a-interferon, a genetically engineered protein, has also shown promise. However, to date there is no known pharmaceutical agent that effectively inhibits the replication of the virus.
To market a nucleoside for pharmaceutical purposes, it must not only be efficacious with low toxicity, it must also be cost effective to manufacture. An extensive amount of research and development has been directed toward new, low cost processes for large scale nucleoside production. 2xe2x80x2,3xe2x80x2-Dideoxynucleosides are currently prepared by either of two routes: derivatization of an intact nucleoside or condensation of a derivatized sugar moiety with a heterocyclic base. Although there are numerous disadvantages associated with obtaining new nucleoside analogues by modifying intact nucleosides, a major advantage of this approach is that the appropriate absolute stereochemistry has already been set by nature. However, this approach cannot be used in the production of nucleosides that contain either nonnaturally occurring bases or nonnaturally occurring carbohydrate moieties (and which therefore are not prepared from intact nucleosides), such as 1,3-oxathiolane nucleosides and 1,3-dioxolane nucleosides.
When condensing a carbohydrate or carbohydrate-like moiety with a heterocyclic base-to form a synthetic nucleoside, a nucleoside is produced that has two chiral centers (at the C1xe2x80x2and C4xe2x80x2-positions), and thus exists as a diastereomeric pair. Each diastereomer exists as a set of enantiomers. Therefore, the product is a mixture of four enantiomers.
It is often found that nucleosides with nonnaturally-occurring stereochemistry in either the C1xe2x80x2or the C4xe2x80x2-positions are less active than the same nucleoside with the stereochemistry as set by nature. For example, Carter, et al., have reported that the concentration of the (xe2x88x92)-enantiomer of carbovir (2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxyguanosine) in cell culture required to reduce the reverse transcriptase activity by 50% (EC50) is 0.8 xcexcM, whereas the EC50 for the (+)-enantiomer of carbovir is greater than 60 xcexcM. Antimicrobial Agents and Chemotherapy, 34:6, 1297-1300 (June 1990).
PCT International Publication No. WO 91/11186 discloses that 1,3-oxathiolane nucleosides can be prepared with high diastereoselectivity (high percentage of nucleoside with a xcex2 configuration of the bond from the C1xe2x80x2-carbon to the heterocyclic base) by careful selection of the Lewis acid used in the condensation process. It was discovered that condensation of a 1,3-oxathiolane nucleoside with a base occurs with almost complete xcex2-stereospecificity when stannic chloride is used as the condensation catalyst. Other Lewis acids provide low (or no) C1xe2x80x2-xcex2 selectivity or simply fail to catalyze the reactions.
In light of the fact that acquired immune deficiency syndrome, AIDS-related complex, and hepatitis B virus have reached epidemic levels worldwide, and have tragic effects on the infected patient, there remains a strong need to provide new effective pharmaceutical agents to treat these diseases that have low toxicity to the host.
There is also a need to provide a cost effective, commercially viable method to produce pharmaceutically important nucleosides, and specifically attain xcex2-stereospecificity in the C4xe2x80x2-position of synthetic nucleosides prepared by condensing a carbohydrate-like moiety with a base.
Therefore, it is an object of the present invention to provide a method and composition for the treatment of human patients infected with HIV.
It is another object of the present invention to provide a method and composition for the treatment of human patients or other host animals infected with HBV.
It is still another object of the present invention to provide enantiomerically enriched 1,3-oxathiolane nucleosides.
It is still another object of the present invention to provide a method for the resolution of C4xe2x80x2-enantiomers of 1,3-oxathiolane nucleosides.
A method and composition for the treatment of HIV and HBV infections in humans and other host animals is disclosed that includes administering an effective amount of 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, a pharmaceutically acceptable derivative thereof, including a 5xe2x80x2or N4 alkylated or acylated derivative, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.
It has been discovered that 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (xe2x80x9cFTCxe2x80x9d), exhibits surprisingly high activity against human immunodeficiency virus with very low host cell toxicity. It has also been discovered that FTC exhibits very significant activity against HBV, and therefore can be used to treat patients who have a variety of illnesses associated with HBV infection.
Toxicity and pharmacokinetic studies confirm the usefulness of FTC as an antiviral agent for pharmaceutical administration. FTC and its enantiomers are nontoxic to peripheral human bone marrow cells at concentrations up to 50 xcexcM and other cell lines at concentrations up to 200 xcexcM. FTC-TP is a major intracellular metabolite in PBMC and HepG2 cells. FTC-TP competitively inhibits HIV-1 reverse transcriptase (RT) with a K1, of 0.2 xcexcM using a poly(I)oligo(dC) template-primer. Using sequencing analysis, FTC-TP can be shown to be a potent DNA chain terminator when HIV-RT is used (C-stops).
Chronic treatment with FTC is not toxic to rodents, even at oral doses of 85 mg/kg per day for at least two months. The pharmacokinetics of FTC in rhesus monkeys indicates high oral bioavailability (approximately 73xc2x16%) and a plasma terminal half life of approximately 1.34xc2x10.18 (mean of oral and I.V. administration).
A process for the resolution of a racemic mixture of nucleoside enantiomers, including the racemic mixture of FTC, is also disclosed that includes the step of exposing the racemic mixture to an enzyme that preferentially catalyzes a reaction in one of the enantiomers. The process can be used to resolve a wide variety of nucleosides, including pyrimidine and purine nucleosides that are optionally substituted in the carbohydrate moiety or base moiety. The process can also be used to resolve nucleoside derivatives that contain additional heteroatoms in the carbohydrate moiety, for example, (xc2x1)-FTC and (xc2x1)-BCH-189. The resolution of nucleosides can be performed on large scale at moderate cost.
Using methods described herein, FTC was resolved into its (+)-xcex2-D and (xe2x88x92)-xcex2-L enantiomers. The (xe2x88x92)-xcex2-L-enantiomer appears to be more potent that the (+)-xcex2-D-enantiomer against HIV, HBV, and SIV. The (+)-enantiomer of FTC is also active against HIV, HBV, and SIV.